The phenomenon of osteoinduction was recognized by Lancroix in 1945 when he demonstrated that acid alcohol bone extracts induced heterotopic bone formation in ectopic sites. Twenty years later Urist and his co-workers decalcified bone matrix and observed new cartilage and bone formation when implanted intramuscularly. These discoveries led to isolation and purification of bone inducing agent named BMPs from bone matrix of different species and years later to cloning and characterization of several cDNAs encoding these novel proteins. The biological activity of BMPs has been determined by bioassay in rat or mouse muscle bounces or by ALP measurements in mammalian cell cultures.
Previous studies since 1965 have shown that BMPs are part of the TGF-β superfamily and like all the family members they have multiple effects on cell migration, growth and differentiation especially in bone formation and tissue repair but also in embryogenesis or cancer. They are low molecular weight hydrophobic glycoproteins which are soluble to chaotrophic agents such as urea and guanidinium hydrochloride but are resistant to several proteases, for example collagenases.
BMPs are produced as large precursor molecules which are processed proteolytically to mature peptides after the translation. Like all the members of TGF-β superfamily, BMPs have the pattern of seven cysteine residues in their C-terminal mature region. Between these cysteines there are three disulfide bonds within mature BMP monomers and one disulfide bond which combines two monomers into a biologically active BMP dimer.
BMPs act through specific transmembrane receptors located on cell surface of the target cells. The BMP receptors are serin-threonin kinases which resemble TGF-β receptors and are divided into two subgroups: type I and type II receptors. BMPs can bind strongly only to the heterotetrameric complex of these receptors. This complex formation is essential to the BMP signal transduction. Inside the target cell, BMP signals are transmitted to the nucleus via specific signal molecules called Smads, which are also responsible for suppression of BMP signals.
Until now, 16 different BMPs have been characterized and seven of them (BMPs 2-7 and 9) have shown to be able to induce bone formation when implanted in ectopic sites. According to the amino acid sequence of the mature part these BMPs are divided into two subgroups. BMPs 2 and 4 are 86% identical and BMPs 5, 6 and 7 are 78% identical. Between these two groups the identity is only about 56%. The amino acid sequence of BMP-3 is about 45% alike with BMPs 2 and 4 and BMP-9 is 50-55% identical with BMPs 2, 4, 5, 6 and 7. Due to high homology and small variety in size, BMPs are quite difficult, very time consuming and expensive to separate, purify and identify from each other at protein level. This is the reason why most of the BMPs are nowadays being produced using molecular biological tools. Different kinds of recombinant protein techniques have been tested and both eukaryotic and prokaryotic systems have been utilized.
Majority of research has focused on human recombinant BMPs, but with regard to effective bone induction antlers of Cervidae family form an interesting research area. Antlers are bony cranial organs typical to the Cervidae family and they differ from Bovidae horns in their growing pattern. Antlers grow from the tip and males cast them away once per year. It has been suggested that antlers are the fastest growing structures through the mammalian species and they are known to be the only structures that regenerate completely every year. Antlers are formed by modified endochondral ossification meaning that the process is performed through the highly vascularized cartilage model which is calcified and finally transformed into bone. Antlers form an interesting model of adult regenerating mineralized tissue, and bone remodeling has been shown to continue until the time of antler casting. Although the ultimate reason for the amazing speed of antler growth has not yet been resolved, antlers have been shown to contain several BMPs. Deer antler has been proven to express BMP-2 and BMP-4 (Feng et al 1997 Biochim Biophys Acta 1350:47-52; Feng et al. 1995 Biochim Biophys Acta 1263:163-168). In addition, reindeer antlers express BMP-3b (Kapanen et al 2002 J Biomed Mat Res 59:78-83). Yet, it is also possible that there is one or more totally uncovered factor(s) which are responsible for antler growth speed.
Due to their osteoinductive capacity, both BMPs extracted from demineralized bone matrix and BMPs produced by recombinant technique are very interesting and highly potential alternatives to bone grafting. Different BMPs have been used in many experimental and clinical studies.
Bone morphogenetic protein 6 has been characterized from many different origins including some mammalian species like mouse, rat, human and bovine. Yet, it has not been characterized from deer animals unlike BMP-2, BMP-3b and BMP-4 which all have been cloned from antlers of either deer or reindeer, both members of Cervidae family (Feng et al 1997; Feng et al 1995; Kapanen et al 2002). Considering the fact that BMP-6 is an important regulator molecule in chondrogenesis it is probable that it is expressed in antlers just as the earlier cloned members of BMP family.
Until now, BMP-3b has been the only BMP characterized in reindeer antler tissue (Kapanen et al 2002).
U.S. Pat. No. 5,399,677 discloses DNA molecules encoding mutant forms of bone morphogenetic proteins. The mutant forms of BMP can be produced bacterially and refolded to produce biologically active homodimers or heterodimers of BMP. A method of making such mutant BMP is also disclosed. Said mutant forms are useful since they are correctly folded when produced in bacterial hosts.
WO 98/51354 discloses osteogenic devices and methods of use thereof for repair of bone and cartilage defects. The method for producing new bone growth at bone defect site in a mammal comprises the step of implanting in a defect site a calcium phosphate matrix comprising at least one osteogenic protein. Said osteogenic proteins include several morphogens, such as bone morphogenetic proteins.
U.S. Pat. No. 6,207,813 discloses purified human BMP-6 proteins and processes for producing them. The publication also discloses commonly known pharmaceutical compositions, medical uses and methods employing said human BMP-6 protein. BMP-6 has been localized in hypertrophic cartilage and it is an important regulator in chondrocyte maturation process. When mouse BMP-6 overexpressing CHO cells were introduced directly into the subcutaneous tissue of athymic nude mice, they formed tumors surrounded by extensive connective tissue containing large regions of cartilage and bone. This suggests that BMP-6 induces endochondral bone formation in vivo. BMP-6 has also been indicated to enhance osteoblastic differentiation of pluripotent mesenchymal progenitor cells in vitro when the cells were transfected with vector overexpressing mouse BMP-6. Furthermore, recombinant human BMP-6 has been shown to take part in osteoblastic differentiation of bone marrow stromal cells in vitro. The osteoinductive activity of recombinant human BMP-6 produced in CHO cells has been verified by in vivo test in rat muscle. Nevertheless, the biological activity of BMP-6 from any origin produced as recombinant protein in E. coli system has not yet been published. Neither has anybody published information of the biological activity of BMP-6 cloned from the antler tissue of any member of Cervidae family.
EP 1131087 discloses further use for morphogenetic proteins, such as BMP proteins. It is shown that exposing cancer cells to morphogens inhibits cancer cell growth and causes such cells to differentiate away from the cancerous phenotype. The use of morphogen can influence cancer cell fate and, in turn, alleviate the symptoms of cancer. Preferred morphogens disclosed include BMP-6.
WO 90/11366 discloses BMP proteins including a bovine BMP-6 for which DNA and amino acid sequences are presented. The amino acid sequence does not correspond to the one currently known for bovine BMP-6 but instead it is identical to the reindeer BMP-6 disclosed herein. The activity or other properties of said BMP-6 are not tested or described in WO 90/11366.
Although some applications of known BMP proteins as bone and cartilage forming inducers or for alleviating the symptoms of cancer are already known, there is still need for better methods for isolating such proteins and for better morphogenetic proteins, for example ones which possess more efficient bone forming properties or are more soluble. Such proteins would be useful for better therapeutic methods and applications. Also methods for producing such proteins would be useful.